SNP ministers requested urgent talks last month after Jose Manuel Barroso, the European Commission president, said a separate Scotland would have to apply from scratch and negotiate the terms of its membership.

But Maros Sefcovic, the commission’s vice-president for inter-institutional relations, refused the request because there is no definite plan for Scotland becoming an independent country.

In a letter to Nicola Sturgeon, Mr Salmond’s deputy, he said the commission was only willing to discuss a “precise scenario” with UK, but not Scottish ministers, ahead of the 2014 independence referendum.

The refusal blunted the First Minister’s claim that David Cameron’s decision to hold a later referendum on the UK’s EU membership was causing uncertainty.

Mr Salmond said it had “completely changed” the independence debate because the largest threat to Scotland’s EU status now came from Westminster and not his separation plans.

In his letter, Mr Sefcovic said the commission did not want to comment on the hypothetical situation of Scottish independence because it is “acutely sensitive to and fully respects the domestic nature of this debate.”

“The European Commission would only be able to express its opinion on the legal consequences under EU law of a specific situation upon request from a Member State detailing a precise scenario,” he wrote.

“At the present stage, in the absence of a precise scenario, President Barroso has, therefore, asked me to signal that he would not be in a position to usefully discuss this further with you.”

This raises the prospect that discussions could take place later in the process, after a Referendum Bill containing the question and date is passed by the Scottish Parliament.

But senior commission sources said Mr Barroso’s stance still held and Scotland would have to apply from scratch. They could not guarantee talks with Scottish ministers will take place before the independence referendum.

Miss Sturgeon said the reply was confirmation the commission has no official view on a separate Scotland’s EU status.

Uncertainty over a separate Scotland’s EU status has dominated the early days of the independence referendum debate and the SNP was quick to seize on Mr Cameron’s promise of a later “in or out” vote on Europe.

Mr Salmond said that by trying to portray himself as a European reformer while keeping his Eurosceptic backbenchers happy, the Prime Minister was “trying to ride two horses at the same time and it is inevitable he will fall off before long”.

“This completely changes the nature of the debate in Scotland. It is now clear the persistent undercurrent of Tory Euro scepticism poses the biggest threat to Scotland's position in the EU,” he said.

But Johann Lamont, the Scottish Labour leader, said: “The SNP and the Tory Party now share the same destructive agenda. They both want referendums which, as Nicola Sturgeon has admitted, cause uncertainty and cost jobs at a time of economic crisis.”